Osteomyelitis is a devastating bone infection, the treatment of which requires a complicated, interdisciplinary approach. This most often involves intensive, long-term systemic antibiotic therapy and surgical debridement accompanied by additional local antibiotic delivery aimed at overcoming the intrinsic resistance of these life- threatening infections while avoiding systemic toxicity. The leading cause of osteomyelitis and other forms of orthopaedic infection is Staphylococcus aureus. Our hypothesis is that overcoming the growing problem of S. aureus orthopaedic infections will require a clear understanding of the bacterial virulence factors that contribute to the development, maturation, persistence, and therapeutic recalcitrance of these infections, as well as the impact these factors have on host bone cell physiology. Our results demonstrate that a key element in this regard is the functional status of the staphylococcal accessory regulator (sarA) and the saeRS regulatory system relative to each other. Specifically, we have established that the functional status of these loci relative to each other can be used to define a virulence gradient in hematogenous osteomyelitis that is defined by the balance between the production of specific virulence factors and their protease-mediated degradation, with the former mediated primarily by the functional status of saeRS and the latter mediated primarily by the functional status of sarA. In this proposal, we will use mutants generated in a contemporary clinical isolate of the USA300 clonal lineage (LAC) that differ in the functional status of these two regulatory loci to fully interrogate this hypothesis in validated animal models of acute hematogenous osteomyelitis, acute post-surgical orthotopic osteomyelitis, and chronic osteomyelitis. This will position us to identify specific virulence factors whose abundance varies in a manner consistent with the virulence gradient defined in vivo, thereby allowing us to identify and prioritize the S. aureus virulence factors that are likely to play important roles in all stages of the osteomyelitis disease process. We will then determine the role of these virulence factors in vivo and investigate the mechanistic basis by which these virulence factors impact host bone cell physiology. Collectively, completion of the proposed studies will set the stage for the development of novel strategies that can be used for the prevention and treatment of S. aureus orthopaedic infections.